The present invention relates to a microfluidic device comprising a microfabricated fluid delivery system and a chromatography column. In particular, the present invention relates to a microfluidic device comprising an OTLC column, PCLC column, or combinations thereof, which is operatively interconnected to a microfabricated fluid delivery system.
Microfluidic devices allow manipulation of extremely small volumes of liquids, and therefore are particularly useful in small scale sample preparations, chemical synthesis, sample assay, sample screening, and other applications where a micro-scale amount of samples are involved. For many applications, the chemical make up of the resulting material (i.e., sample) needs to be analyzed. Such analysis typically requires at least some degree of sample purification and/or separation. However, due to the small sample size (e.g., nanoliter to microliter) used by these microfluidic devices, conventional separation techniques are not applicable.
Use of packed capillary and open tubular liquid chromatography (PCLC and OTLC, respectively) separation techniques have become increasingly popular due to the demonstrated means of achieving high chromatography efficiency with low operation pressures. Conventional high performance liquid chromatography (i.e., HPLC) typically requires  greater than 2000 psi pressure. In contrast, pressure of as low as 5 psi can be used for OTLC and PCLC. Some of the advantages of the OTLC and PCLC techniques include, but are not limited to: (1) an increased efficiency, (2) a lower sample dilution requirement, thereby increasing the sample detection sensitivity, e.g., using a mass spectrometer, (3) a smaller amount of eluent requirement, and (4) the small sample amount requirement. The latter advantage is of particularly importance in a variety of fields, such as proteomics, genomics, forensics, and other areas where a minute quantity of sample is to be separated or purified. Unfortunately, in order to achieve the desired sensitivity and efficiency in OTLC and PCLC, the inner diameter of OTLC and PCLC columns need to be small, generally in the order of 50 xcexcm or less, and preferably about 10 xcexcm or less. The small column diameter size in OTLC and PCLC techniques requires an equally precise sample injection and pumping system. To be effective, OTLC and PCLC techniques require a sample flow rate of 0.01 xcexcL/min or less. Conventional sample pumping system can not adequately meet this stringent requirement. In addition, difficulties with large interconnection dead volume and detection volume between the OTLC or PCLC column and the fluid delivery (i.e., pumping) system have greatly limited the application of OTLC and PCLC techniques.
Therefore, there is a need for OTLC and PCLC devices which comprise a sample injection and fluid pumping system that can achieve a sample flow rate of 0.01 xcexcL/min or less. There is also a need for OTLC and PCLC devices which have small or no dead volume between the OTLC or PCLC column and the fluid delivery system.
One aspect of the present invention provides a microfluidic chromatography apparatus for separating an analyte in a sample fluid. The microfluidic chromatography apparatus of the present invention comprises a microfabricated fluid delivery system and a chromatography column. The microfabricated fluid delivery system of the present invention is capable of pumping a minute amount of fluid through the chromatography column. Preferably, the microfabricated fluid delivery system is capable of pumping (i.e., delivering or transporting) a fluid through the chromatography column at a flow rate of 0.01 xcexcL/min or less. Thus, microfluidic chromatography apparatuses of the present invention are particularly useful in separating analyte(s) from a minute quantity of sample fluid.
Preferably, the fluid delivery system of the present invention is produced from a material comprising an elastomeric polymer. In one particular embodiment, the elastomeric polymer is selected from the group consisting of poly(carborane-siloxanes), poly(bis(fluoroalkoxy)phosphazene), poly(acrylonitrile-butadiene), poly(1-butene), poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers, poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidene fluoride-hexafluoropropylene) copolymer, elastomeric polyvinylchloride, polysulfone, polycarbonate, polymethylmethacrylate, polytertrafluoroethylene, polydimethylsiloxane, polydimethylsiloxane copolymer, and aliphatic urethane diacrylate.
The fluid deliver system of the present invention comprises:
(i) a microfluidic flow channel comprising a flow channel inlet for introducing the fluid into said flow channel and a flow channel outlet,
(ii) a flow control channel,
(iii) a flow control valve comprised of a flow control elastomeric segment that is disposed in between said flow channel and said flow control channel to regulate fluid flow through said flow channel, wherein said flow control valve is deflectable into or retractable from said flow channel upon which said flow control valve operates in response to an actuation force applied to said flow control channel, said flow control elastomeric segment when positioned in said flow channel restricting fluid flow therethrough, and
(iv) a flow control channel actuation system operatively interconnected to said flow control channel for applying an actuation force to said flow control channel.
The fluid delivery system of the present invention can further comprise other component(s) depending on a particular need. For example, in one particular embodiment, the fluid delivery system further comprises a peristaltic pump which is comprised of one or more of the flow control valves.
The fluid delivery system can also comprise an eluent inlet which is in fluid communication with the flow channel inlet for introducing an eluent to said flow channel. In one specific embodiment, the eluent inlet further comprises:
an eluent reservoir comprising an eluent reservoir inlet channel;
an eluent reservoir inlet control channel;
an eluent reservoir inlet control valve for opening and closing fluid communication between said eluent reservoir and said flow channel, wherein said eluent reservoir inlet control valve comprises an elastomeric segment of said eluent reservoir inlet control channel that is disposed in between said eluent reservoir inlet control channel and said eluent reservoir inlet channel to regulate fluid flow through said eluent reservoir inlet channel, wherein said eluent reservoir inlet control valve is deflectable into or retractable from said eluent reservoir inlet channel upon which said eluent reservoir inlet control valve operates in response to an actuation force applied to said eluent reservoir inlet control channel, said elastomeric segment of said eluent reservoir inlet control valve when positioned in said eluent reservoir inlet channel restricting fluid flow therethrough;
an eluent reservoir inlet control channel actuation system operatively interconnected to said eluent reservoir inlet control channel for applying an actuation force to said eluent reservoir inlet control channel.
The flow channel inlet of the fluid delivery system can also comprise:
a sample reservoir comprising a sample reservoir inlet channel which is in fluid communication with said flow channel;
a sample reservoir inlet control channel;
a sample reservoir inlet control valve for opening and closing fluid communication between said sample reservoir and said flow channel, wherein said sample reservoir inlet control valve comprises an elastomeric segment of said sample reservoir inlet control channel that is disposed in between said sample reservoir control channel and said sample reservoir inlet channel to regulate fluid flow through said sample reservoir inlet channel, wherein said sample reservoir inlet control valve is deflectable into or retractable from said sample reservoir inlet channel upon which said sample reservoir inlet control valve operates in response to an actuation force applied to said sample reservoir inlet control channel, said elastomeric segment of said sample reservoir inlet control channel when positioned in said sample reservoir inlet channel restricting fluid flow therethrough; and
an sample reservoir inlet control channel actuation system operatively interconnected to said sample reservoir inlet control channel for applying an actuation force to said sample reservoir inlet control channel.
The chromatography column of the present invention comprises:
(i) a stationary phase which is capable of separating at least a portion of the analyte from the sample fluid,
(ii) a column inlet which is in fluid communication with said flow channel outlet, and
(iii) a column outlet through which a separated fluid exits the chromatography column.
Preferably, the chromatography column is a separately fabricated component which is then integrated with the microfabricated fluid delivery system. Advantages of this embodiment include the capability of using the microfabricated fluid delivery system with a variety of different chromatography columns and interchangeability of chromatography columns depending on the need. Thus, in one particular embodiment, the chromatography column is a microfluidic chromatography device comprising a chromatography channel having an inner surface. Preferably, the stationary phase is covalently bonded to the inner surface of the chromatography channel. The stationary phase can be bonded to the chromatography column by a variety of means conventionally known to one skilled in the art. Such methods include activating or depositing ions on the inner surface of the column. Preferably, the stationary phase is bonded to the inner surface of the column without the need for any surface activation process. In this manner, an integrated microfluidic chromatography system can be fabricated.
In one embodiment, the chromatography column comprises a microfabricated rotary channel comprising:
a rotary channel inlet;
a rotary channel outlet;
a rotary control channel;
a rotary inlet control valve comprised of an elastomeric segment of said rotary inlet control channel that is disposed in between said rotary channel inlet and said rotary control channel to regulate fluid flow into said rotary channel, wherein said rotary inlet control valve is deflectable into or retractable from said rotary channel inlet upon which said rotary inlet control valve operates in response to an actuation force applied to said rotary control channel, said elastomeric segment of said rotary inlet control channel when positioned in said rotary channel inlet restricting fluid flow therethrough;
a rotary outlet control valve comprised of an elastomeric segment of said rotary outlet control channel that is disposed in between said rotary channel outlet and said rotary control channel to regulate fluid flow out of said rotary channel, wherein said rotary outlet control valve is deflectable into or retractable from said rotary channel outlet upon which said rotary outlet control valve operates in response to an actuation force applied to said rotary control channel, said elastomeric segment of said rotary control channel outlet when positioned in said rotary channel outlet restricting fluid flow therethrough;
a rotary pump valve comprised of an elastomeric segment of said rotary pump that is disposed in between said rotary channel and said rotary pump control channel to regulate fluid flow through said rotary channel, wherein said rotary pump valve is deflectable into or retractable from said rotary channel upon which said rotary pump valve operates in response to an actuation force applied to said rotary pump control channel, said elastomeric segment of said rotary pump when positioned in said rotary channel restricting fluid flow therethrough; and
a rotary control channel actuation system operatively interconnected to said rotary control channel for applying an actuation force to said rotary control channel.
In one particular embodiment, the chromatography column is an open tubular liquid chromatography column or a packed capillary liquid column or a combination of these two columns.
The column outlet can also be in fluid communication with a sample detection system inlet. In this manner, the fluid exiting the chromatography column can be analyzed directly with a detection apparatus.
Furthermore, other components, such as sample preparation and detection components, can be fabricated or incorporated within the microfluidic chromatography apparatus of the present invention to provide parallel-processing systems.
In one embodiment of the present invention, the flow channel is located on an interface between a solid substrate and the elastomeric polymer such that an inner surface of the flow channel comprises an elastomeric polymer portion and a solid substrate portion. In one particular embodiment, the stationary phase is attached to the solid substrate portion of the flow channel inner surface. In one specific embodiment, the elastomeric polymer portion of the flow channel inner surface comprises a surface coating that reduces a non-specific binding of the analyte.
Another aspect of the present invention provides a method for producing the microfluidic chromatography apparatus. In one particular embodiment, such a method comprises:
(a) producing a microfabricated fluid delivery system from a material comprising an elastomeric polymer, wherein the fluid deliver system comprises:
(i) a microfluidic flow channel comprising a flow channel inlet for introducing the fluid into said flow channel and a flow channel outlet,
(ii) a flow control channel,
(iii) a flow control valve comprised of a flow control elastomeric segment that is disposed in between said flow channel and said flow control channel to regulate fluid flow through said flow channel, wherein said flow control valve is deflectable into or retractable from said flow channel upon which said flow control valve operates in response to an actuation force applied to said flow control channel, said flow control elastomeric segment when positioned in said flow channel restricting fluid flow therethrough, and
(iv) a flow control channel actuation system operatively interconnected to said flow control channel for applying an actuation force to said flow control channel; and
(b) connecting the fluid delivery system to a chromatography column having a column inlet and a column outlet such that the column inlet is in fluid communication with the flow channel outlet, wherein the chromatography column comprises a stationary phase which is capable of separating at least a portion of the analyte in the fluid.
In addition, methods for producing the microfluidic chromatography apparatus can further include
(a) microfabricating the chromatography column which comprises a chromatography channel having an inner surface which comprises a functional group; and
(b) attaching a stationary phase compound to at least a portion of the inner surface by reacting the stationary phase compound with the functional group under conditions sufficient to form a covalent bond between the functional group and the stationary phase compound.
In one particular embodiment, the functional group is silane.
In another embodiment, the stationary phase compound is 1-octadecene.
The method can also include microfabricating a rotary channel described above.
Yet another aspect of the present invention provides a method for separating an analyte from a sample fluid comprising:
(a) introducing the sample fluid into a microfluidic chromatography a apparatus described above, and
(b) eluting the sample fluid through the chromatography column with an eluent to separate at least a portion of the analyte.
In one particular embodiment, fluid flow through the chromatography column is achieved by a peristaltic pump action created by actuating one or more of the flow control valves.
When the chromatography column comprises a microfabricated rotary channel, the method can further include:
introducing at least a portion of the sample fluid into the rotary channel;
closing the rotary inlet and the rotary outlet control valves by actuating the rotary inlet and the rotary outlet control valves;
transporting the sample fluid through the rotary channel by actuating one or more of the rotary pump valves until at least a portion of the analyte is adsorbed onto the stationary phase;
opening the rotary inlet and rotary outlet control channels;
introducing a first eluent through the rotary inlet channel and removing the resulting mixture through the rotary outlet channel, whereby substantially all of the sample fluid is removed from the rotary channel and at least about 95% of the adsorbed analyte remains adsorbed onto the stationary phase; and
introducing a second eluent, which is capable of removing the analyte from the stationary phase, through the rotary inlet channel and removing the resulting mixture through the rotary outlet channel, whereby substantially all of the adsorbed analyte is removed from the rotary channel.
Such rotary channel chromatography column is particularly useful in separating a large molecules such as proteins and oligonucleotides. In one particular embodiment, the analyte is a protein having a molecular weight of at least about 1000 g/mol. Suitable stationary phases for proteins and oligonucleotides are well known to one skilled in the art. For example, proteins in aqueous solution can be separated using C-18 alkyl as the stationary phase. In this manner, the first eluent is selected from the group consisting of water and an aqueous buffer solution, which removes the sample fluid but substantially leaves the adsorbed proteins bound to the solid phase. By using a second eluent which comprises an organic solvent selected from the group consisting of an alcohol, acetonitrile, dimethylformamide, and mixtures thereof, one can then remove the protein from the stationary phase. The second eluent can also be a mixture of the organic solvent and water or an aqueous buffer solution.